[18F]FLT is a radioactive tracer developed in recent years and used in the PET diagnostic procedure in the field of oncology. Its usefulness lies in its ability to differentiate between benign and malignant tissues, in the possibility of measuring the aggressiveness of the tumour and in being able to evaluate the response to antitumoral therapies at an early step.
Despite the considerable interest in this compound, the number of centres equipped to carry out this procedure is extremely limited, as is the number of studies performed on the topic. The reason lies in the current lack of a method of synthesis of [18F]FLT extremely simple and effective and, above all, which can be easily automated, unlike the case of the radioactive tracer [18F]FDG which, for this and other reasons, is routinely used in most PET centres worldwide.
FIG. 1 shows a known procedure which is used for the preparation of the radioactive tracer 3′-deoxy-3′-[18F] fluorothymidine ([18F]FLT).
In the case in question, the precursor 3-N-Boc-1-[5-O-(4,4′-dimethoxytrityl)-3-O-nitrophenylsulfonyl-2-deoxy-β-D-lyxofuranosyl] thymidine is used, as recently reported in the literature.
The introduction of [18F] fluoride and the subsequent removal of the protective groups with HCl are also well known and commonly used procedures.
The published procedures basically differ in the conditions in which the reactions are carried out (temperature, time, concentration, volume) and in the presence, if any, of an intermediate purification step before the hydrolysis reaction.
The final purification step represents the weak point of these procedures, since HPLC is often used (costly, cumbersome, laborious to manage in routine preparations, more difficult to automate) and must be followed by a “formulation” step (removal of the organic solvent and redissolution of the residue in water or saline solution).
The radiochemical yield is generally low and, more importantly, the end product does not always have the necessary requirements to allow it to be injected in humans. This is due to a number of reasons such as an excessive amount of residual solvents and, if HPLC purification is not carried out, insufficient radiochemical purity and the presence of chemical impurities.
The procedure described in the international patent application no. WO-A-2006/133732 includes an intermediate purification step before hydrolysis, consisting of a hydrolysis reaction on solid support, and an unusual final purification step using disposable commercial columns (commonly called “cartridges” or “SepPaks”).
However, it should be pointed out that implementation of this procedure leads to a series of problems such as:                the automation of the process is laborious and in any case requires a greater number of valves than is normally present on an [18F]FDG module;        there are considerable problems of reproducibility due to obstructions in the tubes caused by partially insoluble compounds;        significant amounts of “cold” impurities may be present;        the end product is dissolved in a watery solution containing a percentage of EtOH (15-30%) which is too high for use in humans (the Official Pharmacopoeia states a maximum value of 0.5%); in addition to these limitations, the final radiochemical yield is on average low.        
The publication “Radiosynthesis of 3′-deoxy-3′-[18F]fluorothymidine: [18F]FLT for imaging of cellular proliferation in vivo” Grierson J. R., Shields A. F. Nuclear Medicine Biology 27, 143-156 (2000) describes a relatively complex and laborious procedure which, precisely because of the difficulties that would be caused by its automation, is carried out almost entirely by hand.
This procedure includes the following steps:
a) Preparation of the anhydrous [18F]F− fluoride;
b) Labelling of the precursor;
c) Hydrolysis;
d) Purification.
The result of the procedure is a radiochemical yield of 42% for precursor A and 40% for precursor B, and a radiochemical purity of 97% for precursor A and of 98% for precursor B. Residual solvents can be detected, with a high percentage (15%) of EtOH.
The procedure described in this publication involves a series of problems and disadvantages which limit its use. In particular:                it requires purification by means of HPLC;        it cannot be transferred to a normal module for [18F]FDG;        it is not possible to use amounts of precursor greater than 40-45 mg as they spoil the separation by means of HPLC;        the procedure takes quite a long time to perform.        
The main disadvantage, however, is the impossibility of injecting the end product into humans due to the high percentage of EtOH.
The international patent application PCT no. WO 2005/025519 describes a procedure consisting of the following steps:
a) Preparation of the anhydrous [18F]F− fluoride;
b) Labelling of the precursors;
c) Hydrolysis;
d) Purification.
In this last step, a solution of sodium acetate is added to the mixture in the reactor in order to neutralise the pH of the solution. The sodium acetate solution volume is sufficient to dilute the DMSO of the reaction mixture to 1:7.
The diluted and neutralised mixture is transferred to a C18 cartridge on which a frit (2 μm) is placed which makes it possible to block any precipitate that could clog the C18 cartridge. Since the frit can also become clogged once part of the mixture has passed through, a valve was inserted in the setup, making it possible to bypass the frit and to load the remaining mixture directly into the C18 cartridge. This valve is controlled by a line pressure reader which, as the pressure increases (due to clogging) above a certain limit, activates the valve and directs the flow towards the bypass line.
It can be noted that this bypass certainly leads to a considerable loss of activity (and thus of product) on the frit and in the lines that connect the frit to the system as they remain full of reaction, mixture and cannot be washed. In addition, the commercial synthesis modules do not generally foresee this bypass system controlled by a pressure reader. The DMSO, even if greatly diluted, tends to carry the [18F]FLT with it which will thus end up in the waste, causing a decrease in the final yield.
The C18 cartridge is then washed with 15.5 mL of water which passes into a waste collection tank. The [18F]FLT is then eluted by passing 1 mL of ethanol through the cartridge and into a second container where it is evaporated.
After the ethanol has evaporated, water is added to the reaction mixture which is then transferred to a collection bottle through a cartridge to remove the [18F] fluoride and a sterile and apyrogenic filter. It can be noted that the ethanol evaporation step requires a second container that can be heated; this is rarely available in commercial synthesis modules. In addition, the ethanol certainly carries away impurities from the C18 cartridge which are then transferred to the end product.